1. Field of the Invention
The present invention relates to cell isolation. More specifically, the present invention relates to non-invasive methods of cell retrieval and isolation.
2. Description of the Related Art
It is thought that due to changing demographics, increased exposure to environmental toxins and intervention in the reproductive process, developmental abnormalities may be on the rise. The risk to any pregnant couple of having a live born infant with a chromosomal abnormality or structural defect has been previously estimated to be between 3% and 5%. Because of this considerable risk, much effort has been expended in recent decades to identify pregnancies at risk of chromosomal anomalies and genetic disorders at an early gestational age. The current standard of care involves screening maternal analytes and ultrasound markers, each alone or in combination, to identify at risk pregnancies, followed by referral for definitive diagnostic tests that include amniocentesis and chorionic villous sampling. While the former screening modalities have considerable rates of false positives and false negatives, the latter diagnostic tests are invasive and carry significant risk of fetal loss. Indeed, Mujezinovic et al. conducted a systematic analysis of 45 studies and reported a fetal loss rate of 1.9% for amniocentesis and 2% for chorionic villous sampling. Therefore, the search to develop safer methods to obtain genetic material from the fetus is ongoing and desperately needed.
Another alternative for prenatal diagnosis is preimplantation genetic diagnosis (PGD), which involves screening for chromosome abnormalities or single gene disorders in an embryo prior to implantation. The main advantage is avoidance of elective pregnancy termination, while offering a high likelihood that the fetus will be free of a specific disorder. Although PGD is an attractive method for prenatal diagnosis, it is an adjunct of assisted reproductive technology that requires in vitro fertilization, which has its own risks and high costs. Thus, PGD is not feasible as a universal diagnostic tool for genetic abnormalities in the general population.
Identification of fetal cells in maternal serum has been attempted, but this approach has been hindered by the relative rarity of fetal cells in maternal blood (1 fetal cell per 106-107 maternal cells) and associated difficulties in their isolation and analysis. Overall, the projected clinical efficacy has been disappointing. Nevertheless, recent discovery of fetal nucleic acids in maternal plasma has introduced several new possibilities for noninvasive prenatal screening of chromosomal aneuploidies. Anomalies are revealed after the first ten weeks of gestation by measuring the allelic ratio of single nucleotide polymorphisms in the coding region of the human genome, analysis of DNA fragments with different patterns of DNA methylation between fetal and maternal DNA, enrichment of the fractional concentration of fetal DNA in maternal plasma using physical or chemical methods, and the development of more precise digital polymerase chain reaction (PCR)-based methods for fetal nucleic acid analysis. Specific inheritable diseases could also be diagnosed with fetal DNA, but due to the fragmented nature of circulating cell-free fetal DNA, maternal plasma screening is not considered a reliable approach.
Prior to 13-15 weeks of gestation, it is believed that small areas of erosions allow trophoblast cells to cross the decidua capsularis and reach the uterine cavity. This process becomes less likely after the amniochorionic membrane seals the uterine cavity and the internal cervical os, which is thought to occur at three months of gestation. In 1971, Shettles suggested that during early pregnancy, a similar shedding occurs into the uterine cavity, making chorionic cellular elements from the degenerating villi available in the endocervical canal. The possibility of capturing fetal cells from accessible regions of the reproductive tract suggests new approaches for early prenatal diagnosis. The isolation of fetal cells from the cervix and the endometrial cavity offers an attractive non-invasive alternative for very early (6-14 weeks, possibly as early as 5 weeks) diagnosis. Since its first description, several investigators have reported the feasibility of isolating fetal cells from the cervical mucus or from fluid obtained by lavage of the endometrial cavity with varying degrees of success. The existing literature suggests that the present status of transcervical cell (TCC) sampling in prenatal diagnosis is experimental, but carries excellent potential for both genetic diagnosis and prediction of pregnancy outcome as laboratory methods are refined and standardized.
The ideal method that would reliably yield fetal cells in appreciable quantity should have no negative impact on the ongoing pregnancy and be free from infectious or traumatic complications. It should also be simple to perform and cost effective, with minimal inter-observer variability. A number of techniques have been devised to retrieve TCC samples from the endocervical canal and the endometrial cavity, including smears obtained with cotton swabs or a cytobrush, aspiration of cervical mucus with a catheter, endometrial biopsy with a Pipelle, and lavage of the endocervical canal or the uterine cavity, all with variable levels of success.
At present, the existing literature differs vastly and is often contradictory in projecting the relative efficacy of the currently available methods for retrieving fetal cells. Previously, emphasis has been placed on the feasibility of obtaining fetal cells and establishing their diagnostic utility, rather than a direct comparison of the relative efficacy of the various methods in randomized control trials, as recently reported. It has been noted that the post-collection processing of the TCC samples has tremendous variation from one study to another, which directly affects the yield of useful information. Techniques used to identify the fetal cells and the diagnostic end points (fetal sex vs gene disorders) have also differed, yielding heterogeneous groups for comparison with non-uniform results. Thus, there is a lack of information on well-described techniques for sample collection and analysis, resulting in considerable dependence on the technique and skill of individual operators.
For example, in the landmark 1971 report by Shettles, identification of the Y chromosome was used to determine fetal sex from midcervical mucus samples obtained with cotton swabs. A limitation of using cotton swabs to retrieve TCC samples is the entrapment of cells within the cotton, which may reduce yield. The use of a cytobrush for cervical mucus retrieval or lavage of the endocervical canal with normal saline offers viable alternatives for TCC collection. A cytobrush inserted through the external os to a maximum depth of 2 cm and rotated at least a full turn during removal provides fetal cells in diagnostic quantities. However, other investigators failed to reproduce this success. Aspiration of the endocervical mucus with a single cannula also results in the detection of fetal cells in up to 70% of TCC samples from mothers with male fetuses. Furthermore, Kingdom et al. demonstrated that lavage of the endocervical canal retrieves more trophoblast cells than the cytobrush, and that cytobrush specimens may have a higher incidence of debris and maternal endocervical cells. A more effective method in terms of fetal cell yield is intrauterine lavage (IUL), in which a flexible catheter connected to a syringe filled with normal saline is used to flush the endometrial cavity. IUL and the other methods for TCC sampling are illustrated in an article by Adinolfi and Sherlock.
Human leukocyte antigen (HLA)-G is a class Ib major histocompatability complex protein that is expressed by human extravillous cytotrophoblast cells and is absent in all other uterine and placental cell populations. In 2003, Bulmer et al. employed MAbs against HLA-G to identify cytotrophoblasts cells in TCC samples retrieved by IUL. Cytotrophoblast cells characterized by their large, irregular hyperchromatic nuclei were HLA-G positive and were identified in 12 of 23 (52%) TCC samples. Interestingly, molecular examination of DNA by QF-PCR in HLA-G positive elements collected by laser capture micro-dissection from four of the patients revealed fetal markers, demonstrating the utility of this approach for prenatal genetic diagnosis. The combined immunohistochemical and molecular approach used in this study revealed considerable variation between the samples. The sensitivity of MAb labeling was relatively low even though HLA-G reactivity provides high specificity for identification of fetal-derived trophoblast cells. HLA-G is expressed by extravillous cytotrophoblast cellular elements, but not by syncytial fragments, limiting its ability to identify all fetal cells. The necessity for a set of MAbs reacting exclusively against antigens expressed on specific subpopulations of trophoblast cells will be crucial for an immunohistochemical approach to identify fetal cells comprehensively. More recently, it was demonstrated that extravillous cytotrophoblast cells could be consistently (>95% of specimens) identified using HLA-G as an antigenic marker in TCC specimens collected by cytobrush into a fixative rinse and prepared on microscope slides free of interfering mucus. Slides stained with the same antibody against HLA-G used by Bulmer et al. and counterstained with hematoxylin reveal a small number of antibody-labeled cytotrophoblast cells on a dense background of cervical cell nuclei. Trophoblast frequency was approximately one in two thousand for all pregnancies successfully sampled between gestation weeks six and fourteen, while this value was reduced four to five-fold in specimens retrieved from women with ectopic pregnancy or blighted ovum. These findings suggest that, in addition to genetic testing, information can be gleaned from TCC analysis alerting clinicians to at-risk pregnancies.
The recovery and analysis of fetal cells shed from the placenta into the cervical canal could provide wider availability of prenatal genetic diagnostics to the general patient population. With improvements in the efficacy and safety of trophoblast collection by TCC sampling using the cytobrush, and in the identification and isolation of those cells expressing trophoblast markers, small quantities of fetal DNA could be readily obtained for genetic testing. New sensitive technologies, such as those now under development for analysis of fetal DNA in maternal serum, could yield extensive information about the fetal genome from modest numbers of isolated cells. The ability to procure cytotrophoblast cells by TCC as early as six weeks of gestation could make this vital information available much earlier than current technologies, including the analysis of fetal DNA in maternal serum. It would therefore be useful to develop a non-invasive method for isolated trophoblasts.